Project 1: Anoctamin8 Tethers the Endoplasmic Reticulum and Plasma Membranes to Assemble Ca2+ Signaling Complexes at ER/PM Junctions. Communication and material transfer between membranes and organelles take place at membrane contact sites (MCSs). MCSs between the ER and PM, the ER/PM junction, is the site where the ER Ca2+ sensor STIM1 and the PM Ca2+ influx channel Orai1 cluster. MCSs are formed by tether proteins that bridge the opposing membranes, but the identity and role of these tether in receptor-evoked Ca2+ signaling is not well understood. Here we identified ANO8 as a key tether in the formation of the ER/PM junctions that is essential for STIM1-Orai1 interaction and channel activation at a PI(4,5)P2-rich domain. Moreover, ANO8 assembles all core Ca2+ signaling proteins: the Orai1, PMCA, STIM1, IP3 receptors and SERCA2 at the ER/PM junctions. This controls the efficiency of receptor-stimulated Ca2+ signaling and duration of Orai1 activity to prevent Ca2+ toxicity. These findings reveal the central role of MCSs in determining efficiency and fidelity of cell signaling. These studies are in revision in Nature communication. Future plans for STIM1, Orai1 and TRPC channels: a) Publish the paper!... b) We continue to examine how STIM1 gates the TRPC and Orai1 channels. At this time, we are studying the role of the ER/PM junctions in regulation of TRPC channels. c) We are analyzing mice with targeted deletion of Orai1 embryonically and in adult pancreas and the salivary glands acinar cells to determine the role of acinar cell secretion of antimicrobials in the control of the gut and oral cavity microbiomes. Topic B) Intracellular Ca2+ channels (15% effort) Project 2: Lysosome signaling controls the migration of dendritic cells. In collaboration with Ana-Maria Lennon-Dumnil (INSERM U932 Immunit et Cancer, Institut Curie Paris, France) we found that found the role of the endolysosomal TRPML1 channel in dendritic cells migration. The study showed that upon bacterial sensing, lysosomal calcium is released by the ionic channel TRPML1, which activates the actin-based motor protein myosin II at the cell rear, promoting fast and directional migration. Lysosomal calcium further induces the activation of the transcription factor EB (TFEB), which translocates to the nucleus to maintain TRPML1 expression. The TRPML1-TFEB axis results from the down-regulation of macropinocytosis after bacterial sensing by DCs. Lysosomal signaling therefore emerges as a hitherto unexpected link between macropinocytosis, actomyosin cytoskeleton organization, and DC migration. These studies have been published in Science Immunology. Future plans for TRPML and TPC channels: We are collaborating with Dr. Freichel (University of Heidelberg) to study regulation of lysosomal and secretory granules Ca2+ signaling by a newly discovered transmembrane protein TMEM63a through the regulation of the TPC channels. Topic C) fluid and HCO3- secretion (35% effort) Project 3: Regulation of Cl- signaling and ion transport by IRBIT-mediated recruitment of multiple kinases and phosphatases. IRBIT is a multifunctional protein that controls the activity of IP3 receptors and the transporters NBCe1-B, CFTR and Slc26a6, by an unknown mechanism. IRBIT interacts with the NBCe1-B N terminus autoinhibitory domain, exposing two cryptic Cl- sensing GXXXP sites to confer regulation of NBCe1-B by intracellular Cl- (Cl-in). Here, LC-MS/MS phosphoprotein analysis revealed that IRBIT controls five NBCe1-B phosphorylation sites that determine both NBCe1-B active conformation and regulation by Cl-in. Specifically, IRBIT-dependent combinatorial dephosphorylation of pSer232, pSer233 or pSer235 generated the conformations pSer233/pSer235, pSer232/pSer235 or pSer232/pSer233. The activity of the pSer232/pSer235 form is similar to IRBIT-activated NBCe1-B, but it is insensitive to inhibition by Cl-in. The pSer232/pSer235 form properties were similar to wild-type NBCe1-B, while the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl-in. In addition, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control Ser65 phosphorylation, which reciprocally affected Cl-in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which reciprocally affected Cl-in sensing by the 194GXXXP198 motif. Ser232/Ser233/Ser235 are conserved in all NBCe1 variants. Mutating the serines to alanines resulted in fully active NBCe1 transporters, while mutating the serines to the phosphomimetic aspartates resulted in inhibited transporters, suggesting that Ser232/Ser233/Ser235 determine whether NBC transporters are in active or inactive conformations. These findings reveal how multiple kinase/phosphatase pathways use multiple phosphorylation sites to fine tune a transport function, which have important implications for epithelial fluid and HCO3- secretion. These studies were in revision in Science Signaling. Future plans for fluid and HCO3- secretion: a) We continue to study the impact of using FDA approved CFTR potentiators and correctors to correct the aberrant fluid secretion in mouse models of Sjgrens syndrome and pancreatitis. Preliminary data suggest that correcting pancreatic function by correcting CFTR affect the microbiome and thus we will explore the effect of improved pancreatic and salivary glands secretion on the microbiome and dell function. b) Synergism between signaling pathways, the Ca2+ and cAMP signaling, is the physiological mode of cell signaling that is aberrant in pathology. We are exploring the molecular mechanism of Ca2+ and cAMP signaling synergism and expansion of signaling repertoire by determining how the scaffolding protein IRBIT makes the Ca2+-activated Cl- and HCO3- permeable channel Best2 to became Ca2+ and cAMP regulated and the physiological significance of this regulation.